This invention relates to enzyme-linked assays.
Alkaline phosphatase (EC 3.1.3.1) is a widely used enzyme label for immunoassays and can also be employed for the determination of gene probes.
Other hydrolases have not enjoyed as wide a usage in enzyme-linked assays owing in part to the non-availability of suitable substrates for employment with them in diagnostic procedures. This invention discloses a family of novel substrates for a range of hydrolases, including alkaline phosphatase, which substrates are hydrolysed to produce a prosthetic group for a detector enzyme, thus providing a large amplification of the signal produced by the hydrolase.
A recently developed method for the assay of this enzyme relies on the hydrolysis of NADP.sup.+ in a primary stage and the subsequent cycling, in a secondary stage, of the NAD.sup.+ produced, resulting in the amplification of the signal (Johannsson et al., J. Immun. Methods, (1986), 87, 7-11, incorporated herein by reference). The technique has not as yet enjoyed widespread use on account of the relative instability of the primary substrate and enzymic cross-talk between components which can result in unacceptable background signals.
A recent technique for the sensitive detection of ribonuclease, our GB 2156518A, relies on the production of a prosthetic group or its precursor, from a prosthetogen. Typically in this methodology the primary enzyme produces riboflavin, which is subsequently converted, by use of one or more ancillary enzymes, to FMN or FAD. Either of these prosthetic groups combines with an apoenzyme to produce a catalytically active holoenzyme which in turn catalyses the signal-producing reaction. In one example the FAD converts apoglucose oxidase (EC 1.1.3.4) to the corresponding holoenzyme, which catalyses the oxidation of glucose thereby producing hydrogen peroxide which can be utilised by peroxidase (EC 1.11.1.7) to produce a coloured product (soluble or insoluble) or light (Thorpe and Kricka, Methods Enzymol. (1986), 133, 331-353, incorporated herein by reference). In another example the FAD converts apo-D-aminoacid oxidase (EC 1.4.3.3) to the corresponding holoenzyme which catalyses the oxidation of, for example, D-alanine which again results in the production of hydrogen peroxide. This can be determined colorimetrically (soluble or insoluble product), luminometrically (Decker and Hinkkanen, Methods Enzymol, (1986), 122, 185-192, incorporated herein by reference) or by using a suitable electrical or electronic biosensor.